The invention provides isolated nucleic acid and amino acid sequences of HsKif15, methods of detecting HsKif15 and screening for HsKif15 modulators using biologically active HsKif15, and kits for screening for HsKif15 modulators.
Kinesin defines a ubiquitous, conserved family of over 50 proteins that can be classified into at least 8 subfamilies based on primary amino acid sequence, domain structure, velocity of movement, and cellular function. The kinesin superfamily is an extended family of related microtubule motor proteins. This family is exemplified by xe2x80x9ctruexe2x80x9d kinesin, which was first isolated from the axoplasm of squid, where it is believed to play a role in anterograde axonal transport of vesicles and organelles (see, e.g., Goldstein, Annu. Rev. Genet. 27:319-351 (1993)). Kinesin uses ATP to generate force and directional movement associated with microtubules (from the minus to the plus end of the microtubule, hence it is a xe2x80x9cplus-end directedxe2x80x9d motor). Kinesin superfamily members are defined by a kinesin-like motor that is about 340 amino acids in size and shares approximately 35-45% identity (or more) with the xe2x80x9ctruexe2x80x9d kinesin motor domain. Typically, the motor is attached to a variety of tail domains that provide different binding activities to the various kinesin superfamily members.
Mouse Kif15 (Genbank accession numbers AB001432) was originally identified in a PCR-based search for novel murine kinesins (Nakagawa et al. 1997. Proc Natl Acad Sci U.S. Pat. No. 9,496,549). A portion of the MmKif15 cDNA encoding a fragment of the MmKif15 motor domain was cloned and sequenced. In addition, the mRNA expression of MmKif15 in several tissues from 4 week old mice was examined.
XKlp2 refers to a Xenopus laevis kinesin that has been shown to play an important role in mitotic spindle assembly. XKlp2 was originally identified by Vemos et al. in a PCR-based strategy to clone cDNA fragments encoding novel Xenopus kinesins (Vernos et al. 1993. Dev Biol 157:232-9). The full length sequence of XKlp2 was published by Boleti et al (Boleti et al. 1996. Cell 84:49-59) (Genbank accession numbers B48835, AAB26486, 1587181, and CAA63826). These authors also demonstrated that a recombinant fusion protein containing the motor domain of XKlp2 fused to Glutathione-S-transferase was capable of microtubule-based motility, moving toward microtubule plus ends. Boleti et al found that XKlp2 associates with mitotic spindle poles and with centrosomes, and that addition of recombinant fragments of XKlp2 spanning the C-terminal tail domain caused a dose-dependent inhibition of bipolar spindle assembly in spindle assembly reactions in vitro. Antibodies directed against the C-terminal tail domain of XKlp2 similarly impaired mitotic spindle assembly.
Wittman et al. have demonstrated that the C-terminal tail domain of XKlp2 is necessary and sufficient for localization to microtubule asters assembled in mitotic but not interphase cell extracts (Wittmann et al. 1998. J Cell Biol 143:673-85). This localization required dimerization of the tail domain. In addition, the function of cytoplasmic dynein and dynactin were found to play a role in localization of XKlp2 to the microtubule asters assembled in mitotic cell extracts. Wittman et al also identified a protein, TPX2, that significantly enhanced binding of recombinant XKlp2 tail domain to pure microtubules. It was suggested that TPX2 is the receptor for the tail domain of XKlp2 that mediates interaction with microtubules.
Walczak et al. have examined the role of XKlp2 in formation of bipolar mitotic spindles using mitotic cell extracts and DNA coated magnetic beads (Walczak et al. 1998. Curr Biol 8:903-13). In contrast to the observations of Boleti et al., Walczak et al did not observe signficant perturbation of mitotic spindle function upon addition of antibody directed against XKlp2 C-terminal tail domain.
The discovery of a new kinesin motor protein and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of cancer, neurological disorders, and disorders of vesicular transport.
The present invention is based on the discovery of a new human kinesin motor protein, HsKif15, the polynucleotides encoding HsKif15, and the use of these compositions for the diagnosis, treatment, or prevention of cancer, neurological disorders, and disorders of vesicular transport.
In one aspect, the invention provides an isolated nucleic acid sequence encoding a kinesin superfamily motor protein, wherein the motor protein has the following properties: (i) the protein""s activity includes microtubule stimulated ATPase activity; and (ii) the protein has a sequence that has greater than 70% amino acid sequence identity to SEQ ID NO:2 as measured using a sequence comparison algorithm. In one embodiment, the protein further specifically binds to polyclonal antibodies raised against SEQ ID NO:2.
In one embodiment, the nucleic acid encodes HsKif15 or a fragment thereof. In another embodiment, the nucleic acid encodes SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.
In another embodiment, the nucleic acid has a nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.
In one aspect, the nucleic acid comprises a sequence which encodes an amino acid sequence which has one or more of the following characteristics:
greater than 70% sequence identity with SEQ ID NO:2, preferably greater than 80%, more preferably greater than 90%, more preferably greater than 95% or, in another embodiment, has 98 to 100% sequence identity with SEQ ID NO:2.
In one embodiment, the nucleic acid comprises a sequence which has one or more of the following characteristics:
greater than 55 or 60% sequence identity with SEQ ID NO: 1, preferably greater than 70%, more preferably greater than 80%, more preferably greater than 90 or 95% or, in another embodiment, has 98 to 100% sequence identity with SEQ ID NO: 1. In another embodiment provided herein, the nucleic acid hybridizes under stringent conditions to a nucleic acid having a sequence or complementary sequence of SEQ ID NO: 1.
In another aspect, the invention provides an expression vector comprising a nucleic acid encoding a kinesin superfamily motor protein, wherein the motor protein has the following properties: (i) the protein""s activity includes microtubule stimulated ATPase activity; and (ii) the protein has a sequence that has greater than 70% amino acid sequence identity to SEQ ID NO:2 as measured using a sequence comparison algorithm. The invention further provides a host cell transfected with the vector.
In another aspect, the invention provides an isolated kinesin superfamily motor protein, wherein the protein has one or more of the properties described above. In one embodiment, the protein specifically binds to polyclonal antibodies generated against a motor domain, tail domain or other fragment of HsKif15. In another embodiment, the protein comprises an amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.
In one aspect, the protein provided herein comprises an amino acid sequence which has one or more of the following characteristics:
greater than 70% sequence identity with SEQ ID NO:2, preferably greater than 80%, more preferably greater than 90%, more preferably greater than 95% or, in another embodiment, has 98 to 100% sequence identity with SEQ ID NO:2.
The invention features a substantially purified polypeptide comprising the amino acid sequence of SEQ ID NO:2 or a fragment thereof and more particularly, the motor domain of the amino acid sequence of SEQ ID NO:2.
In another aspect, the invention provides a method for screening for modulators of HsKif 15, the method comprising the steps of: (i) contacting biologically active motor protein having at least one of properties described above, with at least one candidate agent at a test and control concentration and detecting whether a change in the activity of the motor protein occurs between the test and control concentration, wherein a change indicates a modulator of the motor protein. In one embodiment, the activity is selected from the group consisting of microtubule stimulated ATPase activity and microtubule binding activity. In one embodiment, the method further comprises the step of isolating biologically active HsKif15 from a cell sample. In another embodiment, the biologically active HsKif15 is recombinant.
In another aspect, the invention provides a kit for screening for modulators of HsKif15, the kit comprising; (i) a container holding biologically active HsKif15; and (ii) instructions for assaying for HsKif15 activity, wherein the HsKif15 activity is microtubule binding activity or microtubule stimulated ATPase activity.